1. Field of the Invention
This invention relates generally to a baculovirus expression vector and/or a recombinant baculovirus with an engineered baculovirus fp25k gene with an improved resistance to mutation during the process of producing a desired protein, virus, protein hybrid, or virus hybrid.
2. Description of the Related Art
Baculoviruses in the family of Baculoviridae are insect-specific viruses with a circular dsDNA genome of 80-180 kbp. The typical species of the family is Autographa californica multiple nucleopolyhedrovirus_(AcMNPV) that has been extensively studied due to its propensity to replicate in many insect cell lines such as Sf21, Sf9 and High Five (Hi5), which makes the AcMNPV-cell systems particularly useful in baculovirus genetic studies.
The popularity of baculovirus to researchers other than baculovirologists is due to its powerful high exogenous gene expression capability in insect cells commonly known as the baculovirus expression vector systems or BEVS. The high protein expression was first recognized by the formation of large polyhedra of 0.5-15 μm in diameter, consisting of a viral polyhedrin (polh) protein in infected insect cells. The high foreign gene expression yield is recognized by the strong promoter of polh that allows the viral RNA polymerase and some late expression factor (LEF) to initiate transcription at extremely high rates for high foreign gene expression during insect cell infection. Of the many baculoviruses reported, AcMNPV is the first baculovirus that has been used for high yield human beta interferon expression under the control of the strong polh promoter. In addition, for high protein expression, the insect cell lines are also critical. Therefore, the Hi5 cell line was cloned from the embryos of Trichoplusia ni and has been used to support high protein expression yield of some genes that leads to be the choice of expressing the major capsid protein L1 of human papilloma-virus (HPV) by the AcMNPV-based BEVS for the production of a high cost prophylactic Cervarix® vaccine to prevent HPV infection-caused cervical cancer by GlaxoSmithKline Biologicals. In addition to Hi5 cells, Sf21 and Sf9 cell lines (a clone of Sf21), derived from pupal ovaries of Spodoptera frugiperda, have been used for protein expression in research laboratories and industry.
During protein expression in Hi5 cells using the strong polh promoter-based AcMNPV BEVS, the AcMNPV fp25k gene mutates at a high frequency that leads to down-regulating polh-promoter activities thus a reduction of protein expression by up to 70% compared to that without fp25k mutations (Cheng et al., 2013 J. Gen. Virol 94: 166-176; Harrison et al., 1996 Virology 226: 34-46). Accompanied with the reduced polh promoter activities is the enhanced production of virus in insect cell growth media (Harrison and Summer, 1996 Virology 226: 34-46; Kelly et al., 2008 Virus Res. 133: 157-166; Wu et al., 2005 J. Gen. Virol. 86: 2439-2444). Mutations of AcMNPV fp25k are reported due to slippage replication errors of the two 7-adenine mononucleotide repeats (MNR) and a 287 bp host DNA insertion at the 10th TTAA site in Sf21 cells (Cheng et al., 2013 J. Gen. Virol 94: 166-176; Gin et al., 2010 J. Gen. Virol. 91: 3053-3064). Therefore, there are three high mutational “hot spots” in the AcMNPV fp25k gene.